To request permission to reproduce material from this article in a commercial publication, Provided that the correct acknowledgement is given and it is not used for commercial purposes. This article in other publications, without requesting further permission from the RSC, Thomas,Ĭreative Commons Attribution-NonCommercial 3.0 Unported Licence. Identification of novel functional mini-receptors by combinatorial screening of split-WW domains Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.
To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1 WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Furthermore, by using 1H– 15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1 WW in CC-hPin1 WW-L1 were fully restored by CC association. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1 WW-L1, but not in CC-hPin1 WW-L2. Fragmentation of hPin1 WW was performed in both loop 1 (CC-hPin1 WW-L1) and loop 2 (CC-hPin1 WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1 WW) by CC association. Since the sequence-structure relationships of β-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. Β-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications.
0 kommentar(er)
